Bacteriophage (phage) are viruses that specifically infect and lyse bacteria. Phage therapy, a method of using whole phage viruses for the treatment of bacterial infectious diseases, was introduce by Felix d'Herelle, who discovered phage around 1920. In the beginning of the 20th century, there were various studies of the application of phage for therapy in humans as well as in animals. In 1940 Eli Lilly Company produced 7 phage products for human use, including phage preparations for treating different sicknesses caused by Staphylococcus sp., E. coli and other pathogenic bacteria. These preparations were utilized to treat infections that cause abscesses, purulent wounds, vaginitis, acute chronic upper-respiratory tract infections and mastoid infections.
However, with the arrival of antibiotics in the 1940's, the development of phage based therapeutics declined in the Western word. One of the most important factors that contributed to the decline of interest in phage therapy in the Western world was the problem of credibility. The reduction in the number of appropriately conducted studies and the lack of well-established protocols and standardizations interfered with the rigorous documentation of the value of phage therapy. Many problems related to the production of phage samples/specimens also complicated the initial study/research related to phage therapy. Diverse stabilizers and preservatives were used in attempts to increase the viability of the phage therapeutics. However, without a good understanding of the biological nature of phage and their stability in response to various physical and chemical agents, many of the ingredients added to prolong the viability of the phage preparations resulted in a negative effect on the viability of the phage, and in some cases proved to be toxic to humans. Another problem related to phage production was the purity grade of the commercial preparations of these viruses. The phage therapy preparations, including those originating from well-established companies in the United States and other countries, consisted of raw lysates of the host bacteria treated with the phage of interest. Thus, the preparations had bacterial components, including endotoxins, that could have adverse effects in patients treated with these preparations, particularly those receiving intravenous administration. However, the use of bacteriophage for therapeutic ends continued jointly with, or in place of antibiotics, in Eastern Europe and in the former Soviet Union where access to antibiotics was limited.
With the rise of antibiotic resistant strains of bacteria, interest in phage based therapeutics has gained broader interest. Even though novel classes of antibiotics may be developed, the prospect that bacteria will eventually develop resistance to the new drugs has intensified the search for non-chemotherapeutic means for controlling and treating bacterial infections. There are three general strategies for using phage-based therapies in a clinical environment: 1) the use of active, virulent phage; 2) the use of endolysins or purified lysins isolated from bacteriophage; and 3) the use of a structural protein of the identified phage as a metabolic inhibitor of key enzymes for the synthesis of bacterial peptidoglycan.
Among the most promising of the strategies currently in development are phage lysins. Preparations of purified endolysins can be used as therapeutic agents, per se, or combined with classic antibiotics. The addition of exogenous lysins to susceptible gram-positive bacteria can cause a complete lysis in the absence of bacteriophage (Loeffler et al., 2001, Science 294:2170-2172; Shuch et al., 2002, Nature 418:884-889). Microscopic images of bacteria treated with a lysin indicate that these enzymes exercise their lethal effect by digesting peptidoglycan leading to the formation of holes in the cell wall. Compared with the external environment, the inside of a bacterium is hypertonic, and when the bacterial wall loses its structural integrity the result is the extrusion of the cytoplasmic membrane and hypertonic lysis.
While penicillin and antibiotics of the Cephalosporin class inhibit the synthesis of peptidoglycan causing lysis of the bacterial cell wall during cell division, the phage lysins destroy the peptidoglycan directly, exercising their lytic effect seconds after being administered. The lysins can also destroy the cell wall of bacteria that are not growing and are insensitive to many antibiotics. When simultaneously administered, two lysins that have differing target sequences may attack the peptidoglycan in multiple regions, presenting a synergistic effect.
There is a clear need for further investigation of lysin enzymes as potential therapeutic and prophylactic agents of use, in vivo, to eliminate pathogenic bacteria without affecting the normal surrounding flora. Due to serious problems of resistant bacteria in hospitals, particularly Staphylococcus and Pneumococcus sp., these enzymes can be an immediate benefit in these types of environments.
However, most lysins discovered to date are specific to species (or subspecies) of bacteria from which they are produced. For example, it has been shown that lysins isolated from streptococcal phage only kill certain streptococci and that lysins produced by pneumococcal phage only kill pneumococci (Fishcetti, 2005, Trends in Microbio 13:491-496). Therefore, there is an increasing need to discover new and novel lysin enzymes that may be used to treat the increasing number of bacterial species that have developed antibiotic resistance. There is also a need to develop lysin constructs that permit species cross-reactivity. In particular, the isolation and/or development of novel lysins with lytic killing or antibacterial activity beyond the specific species from which they are isolated would be especially valuable.